Báo cáo khoa học: Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies pot

MINIREVIEW
Alternative splicing: role of pseudoexons in human disease
and potential therapeutic strategies
Ashish Dhir and Emanuele Buratti
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Introduction
Towards the end of the 1970s, in the beginning of
pre-mRNA splicing research [1,2], defining exons and
introns was essentially based on observing the final
composition of the mature mRNA molecule. In 1978,
any sequence that was included in a mature mRNA
became tagged as an ‘exon’, whereas all the intervening
genomic sequences that were left out during the splic-
ing process became defined as ‘introns’ [3]. However,
this way of thinking did not explain what makes an
exon an exon or an intron an intron. The discovery of
the basic splice site consensus sequences during the
same years [4,5], and later on of enhancer and repres-
sor elements, has taken us a long way in the direction
of discovering exon- and intron-definition complexes
[6–8]. Nowadays, the splicing signals that define ex-
ons ⁄ introns have been greatly aided by basic research,
bioinformatic approaches and advanced sequencing
tools [9,10]. In this regard, we certainly know much
more about splicing regulation than we did 20 years
ago. Considering that several reviews have been writ-
ten recently on the subject, the reader is referred to
them for further information on the latest discoveries
[11–14]. Most important, in this respect, have been the
initial observations that in alternative splicing pro-
cesses the same nucleotide sequence could be defined

by the spliceosome as an intron or an exon in response
to specific signals [15,16]. It is now clear that these
kinds of decision (What is an exon? What is an
intron?) are of paramount importance in explaining
genome complexity and evolutionary pathways
[17–20]. However, the sum of this new knowledge does
not necessarily mean that we are near the goal of
Keywords
alternative splicing; antisense
oligonucleotides; mRNA; pseudoexons;
splicing therapy
Correspondence
E. Buratti, Padriciano 99, 34012 Trieste, Italy
Fax: +39 040 226555
Tel: +39 040 3757316
E-mail:
(Received 26 August 2009, revised 15
October 2009, accepted 5 November 2009)
doi:10.1111/j.1742-4658.2009.07520.x
What makes a nucleotide sequence an exon (or an intron) is a question
that still lacks a satisfactory answer. Indeed, most eukaryotic genes are full
of sequences that look like perfect exons, but which are nonetheless
ignored by the splicing machinery (hence the name ‘pseudoexons’). The
existence of these pseudoexons has been known since the earliest days of
splicing research, but until recently the tendency has been to view them as
an interesting, but rather rare, curiosity. In recent years, however, the
importance of pseudoexons in regulating splicing processes has been stea-
dily revalued. Even more importantly, clinically oriented screening studies
that search for splicing mutations are beginning to uncover a situation
where aberrant pseudoexon inclusion as a cause of human disease is more

frequent than previously thought. Here we aim to provide a review of the
mechanisms that lead to pseudoexon activation in human genes and how
the various cis- and trans-acting cellular factors regulate their inclusion.
Moreover, we list the potential therapeutic approaches that are being tested
with the aim of inhibiting their inclusion in the final mRNA molecules.
Abbreviations
3¢ss, 3¢ splice site; 5¢ss, 5¢ splice site; AON, antisense oligonucleotide; LINE, long interspersed elements; NMD, nonsense-mediated decay;
PTB, polypyrimidine tract binding protein; SINE, short interspersed elements.
FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS 841
understanding most splicing decisions. Indeed, even
the latest attempts at ‘designing’ exons based on
current state-of-the-art knowledge have basically dem-
onstrated that there is still a long way to go before we
can become as good as the spliceosome in deciding
what is an exon and what is an intron [21].
Where do pseudoexon sequences come
into the story?
Central to the issue of deciding what is an exon and
what is an intron is the question of their origin, a very
much debated field to this day that basically deals with
deciding the order of appearance of introns during
evolution, whether first, early or late [22]. Whatever
the answer to this question will turn out to be, it is
now clear that many of the ‘new’ exons in our genome
originate from the insertion of transposable sequence
elements belonging to the SINE and LINE classes in
the eukaryotic genome [23–25]. In particular, exoniza-
tion of Alu elements (which are primate specific and
represent the most abundant mobile elements in the
human genome) through retrotranposition–mutation

events is a prominent source of new exons in the
eukaryotic transcriptome, as schematically depicted in
Fig. 1 [26,27].
However, even if we ignore this particular class of
exonization event, every in silico analysis shows that
‘false exons’ are very abundant in the intronic
sequences of most genes [with this term we refer to
any nucleotide sequence between 50 and 200–300
nucleotides in length with apparently viable 5¢ and 3¢
splice sites (5¢ss and 3¢ss) at either end]. Presently,
there is evidence that inclusion of many of these
sequences is actively inhibited due to the presence of
intrinsic defects [28], the presence of silencer elements
[29–31] or the formation of inhibiting RNA secondary
structures [32]. Even if a combination of all these ele-
ments succeeds in repressing the use of many of these
pseudoexon sequences, we have to consider the possi-
bility that there must be many exceptions to this rule.
First, it is probable that several of these pseudoex-
ons may actually be recognized only in particular cir-
cumstances, such as a consequence of particular
external stimuli [33,34] or present in a given tissue or
developmental stage. Proof of this possibility is the
observation that ‘novel’ exons keep being identified
even in well-known and studied genes, such as the
DMD gene [35].
Second, our failure to observe their use in normal
conditions may also be due to the fact that their inclu-
sion can intentionally lead to premature insertion of a
termination codon in the mature mRNA and the con-

sequent rapid degradation by nonsense-mediated decay
(NMD) pathways [36] (Fig. 1). Such an occurrence has
been described in the rat a-tropomyosin gene with a
putative pseudoexon sequence localized downstream of
two mutually exclusive exons: an upstream exon that
is included only in smooth muscle tissue and a down-
stream exon that is included in most cell types [37].
Fig. 1. The left panel shows a schematic model of Alu element exonization. The element (Alu) is inserted by retrotransposition and during
the course of evolution mutations within this sequence create viable splicing sequences. The middle panel shows the effect of the inclusion
of a nonsense exon sequence (NE) in a transcript. When this nonsense exon sequence is included, the resulting transcript is degraded by
NMD (lower diagram). The right panel shows the classical pathway of pseudoexon (PE) inclusion in human disease. In this case, a nucleotide
sequence on the brink of becoming an exon becomes activated following a number of different mutational events.
Pseudoexons in human disease A. Dhir and E. Buratti
842 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
Experimental analysis has shown that, when this
pseudoexon is included in the mRNA molecule
together with the ubiquitously expressed downstream
exon, the formation of a stop codon causes activation
of the NMD pathway. On the other hand, when inclu-
sion of this pseudoexon occurs with the upstream
smooth muscle tissue-specific exon, then it can still be
removed through a resplicing pathway (and a normally
processed mRNA molecule can be generated). For this
reason, the term ‘nonsense’ exon is now preferred to
define these kinds of sequence, which according to
bioinformatic analyses may be more prevalent in
human genes than previously thought [37].
Nonetheless, from a human disease point of view,
many pseudoexon intronic sequences seem poised on
the brink of becoming exons (Fig. 1) and a compre-

hensive list of more than 60 published pathological
pseudoexon events is presented in Table 1. Although
briefly reviewed previously elsewhere [38], the recent
advances in pseudoexon research warrant a second
look at several pseudoexon-related issues, especially
with regards to novel therapeutic approaches.
Cis-acting sequences in pseudoexon
inclusion
As previously mentioned, most pathological pseudoex-
on inclusion events originate from the creation of new
splicing donor or acceptor splice sites within an intron-
ic sequence, followed by the subsequent selection
of weaker ‘opportunistic’ acceptor or donor site
sequences (Fig. 2A). A preliminary analysis of the
strength of donor sites activated in pseudoexon inclu-
sion events has highlighted their relatively high
strength (according to in silico prediction programs)
with respect to normally processed exons and to cryp-
tic donor sites activated following normal donor site
inactivation [39]. In a slightly lower number of cases,
pseudoexon activation has been observed following the
creation of de novo acceptor sites (Table 1), whereas
branch-point creation still represents a minority (prob-
ably owing to the fact that a new branch point needs
to find both a viable acceptor and donor site nearby,
rather than just one of them).
In addition to de novo creation of strong donor,
acceptor and branch site sequences, the other most fre-
quent mechanisms that may lead to pseudoexon activa-
tion involves the creation ⁄ deletion of splicing

regulatory sequences that will be discussed more in
detail below (Fig. 2B). Finally, in two individual cases,
the rearrangement of genomic regions through gross
deletions (Fig. 2C) [40] or genomic inversions
(Fig. 2D) [41] has also been described to give rise to
pseudoexon inclusion events. This has come about
either by bringing together viable splice sites that
would normally be too far away from each other on
the gene sequence or by activating exons in what
would normally have been the antisense genomic
strand.
In a few genes, a particularly interesting method of
pseudoexon activation event has also occurred follow-
ing the inactivation of naturally occurring up
stream 5¢ss (FAA, IDS, MUT) [42–45] or downstream
3¢ss (BRCA2, CFTR) [46,47] (Fig. 2E). These findings
suggest that the processivity of these mRNA tran-
scripts probably represents an element capable of
determining pseudoexon repression apart from being
capable of influencing normal splicing levels [48].
On a more general note, a still underappreciated
aspect of pseudoexon recognition that concerns the
effect of cis-acting sequences is represented by the
potential influence of RNA secondary structure on
splicing efficiency [49]. Recently, it has been shown
that donor site usage in the inclusion of two pseudoex-
on sequences in the ATM and CFTR genes is strongly
dependent on their availability in the single-stranded
region [50]. Interestingly, the same conclusion was
reached in a recent study by Schwartz et al. [51] analy-

sing the differences between exonized and nonexonized
Alu elements. In this work, it was found that one of
the major discriminating factors between these two
classes of Alu elements was represented by the poten-
tial availability of 5¢ss sequences in an unstructured
conformation.
Trans-acting factors in pseudoexon
inclusion
Not many studies have focused on identifying the role
played by trans-acting factors in pseudoexon inclusion.
However, because of its significance, this is an area of
research that would probably benefit from increased
attention by researchers in the future.
In the case of nonpathologically related pseudoex-
ons carrying nonsense codons, the presence of splicing
regulatory elements may well provide a clue with
regards to the possible roles played by these
sequences. For example, in the case of the previously
described tropomyosin pseudoexon [37], the specific
binding of hnRNP H ⁄ F proteins has been described
as a potential key modifier of this pseudoexon inclu-
sion event [52]. The fact that these proteins are partic-
ularly downregulated in cardiomyocytes may explain
the cell-specific repression of the downstream ‘normal’
exon 3 that is otherwise present in all cell types
(Fig. 3A).
A. Dhir and E. Buratti Pseudoexons in human disease
FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS 843
Table 1. Pathological pseudoexon inclusion events in human disease. NA, not available; SRE, splicing regulatory element.
Gene Size (bp)

Activating
mutation Reference DBASS3 ⁄ DBASS5 reference
a-Gal A 57 SRE creation [78] http: ⁄⁄www.som.soton.ac.uk ⁄ research ⁄ geneticsdiv ⁄ dbass5 ⁄ viewsplicesite.aspx?id=317
ATM 65 SRE deletion [56] http: ⁄⁄www.som.soton.ac.uk ⁄ research ⁄ geneticsdiv ⁄ dbass5 ⁄ viewsplicesite.aspx?id=324
ATM 137 5¢ss creation [79] />b-globin 165 5¢ss creation [80] />b-globin 126 5¢ss creation [81] />b-globin 73 5¢ss creation [82] />BRCA1 66 3¢ss creation [83] NA
BRCA2 93 Downstream
3¢ss deletion
[46] NA
CD40L 59 5¢ss creation [84] />CEP290 128 5¢ss creation [85] />CFTR 49 5¢ss creation [86] />CFTR 84 5¢ss creation [87] />CFTR 101 SRE creation [88] NA
CFTR 184 Downstream
3¢ss deletion
[47] />CFTR 214 5¢ss creation [89] />CHM 98 3¢ss creation [90] NA
COL4A3
a
74 3¢ss creation [91] NA
COL4A5 30 3¢ss creation [92] />COL4A5 147 SRE creation [92] NA
CTDP1 95 5¢ss creation [93] />CYBB 56 5¢ss creation [94] NA
CYBB 61 5¢ss creation [95] />DHPR ⁄
QDPR
152 5¢ss creation [96] />DMD 98 28 kb gene
inversion
[41] NA
DMD 108 28 kb gene
inversion
[41] NA
DMD 125 28 kb gene
inversion
[41] NA
DMD 149 28 kb gene
inversion

[41] NA
DMD 160 28 kb gene
inversion
[41] NA
DMD 180 28 kb gene
inversion
[41] NA
DMD 58 5¢ss creation [97] />DMD 67 5¢ss creation [98] NA
DMD 89 5¢ss creation [98] />DMD 90 5¢ss creation [98] />DMD 95 3¢ss creation [97] />DMD 147 5¢ss creation [99] />DMD 149 3¢ss creation [98] />DMD 172 ⁄ 202 5¢ss creation [100] />DMD 46 ⁄ 132 3¢ss creation [101] />FBN1 93 5¢ss creation [102] NA
FGB 50 SRE creation [63] NA
FGG 75 5¢ss creation [103] NA
FVIII 191 5¢ss creation [104] />GALC 34 ND [105] />GHER 69 5¢ss creation [106] NA
GHR 102 SRE deletion [57,107] />GUSB
a
68 5¢ss creation [108] />Pseudoexons in human disease A. Dhir and E. Buratti
844 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
Interestingly, repression of the tropomyosin non-
sense exon was also observed following PTB overex-
pression. PTB is a well-known and powerful splicing
modifier that plays a major role in alternative splicing
regulation [8,53]. Recently, this protein has been
reported to also downregulate the inclusion efficiency
of a pathological pseudoexon in NF-1 intron 31 inde-
pendently of the activating mutation that creates a
very strong splicing acceptor site [54] (Fig. 3B). This
finding suggests that silencer binding sites may be
Table 1. (Continued.)
Gene Size (bp)
Activating
mutation Reference DBASS3 ⁄ DBASS5 reference

HADHB 56 ⁄ 106 5¢ss creation [109] NA
HSPG2 130 5¢ss creation [110] />IDS 78 5¢ss creation [111] />IDS 103 Upstream 5¢ss
deletion
[42,43] />INI1 ⁄
SNF5
72 5¢ss creation [112] />ISCU 86 ⁄ 100 3¢ss creation [113–115] NA
JK 136 Internal 7 kb
deletion
[40] />MCBB 64 SRE deletion [116] NA
MYO6 108 5¢ss creation [117] NA
MUT 76 5¢ss creation
or upstream
5¢ss deletion
[45,68] /> />NDUFS7 122 5¢ss creation [118] />NF-1 70 5¢ss creation [70] NA
NF-1 107 5¢ss creation [70] NA
NF-1 172 3¢ss creation [119] />NF-1 58 3¢ss creation [120] NA
NF-1 76 5¢ss creation [120] NA
NF-1 54 5¢ss creation [121] />NF-1 177 5¢ss creation [70,122,123] />NF-2 106 Branch-point
creation
[124] NA
NPC1 194 5¢ss creation [125] NA
OA1 ⁄
GPR143
165 3¢ss creation [69] />OAT
a
142 5¢ss creation [126] />OTC 135 3¢ss creation [127] NA
PCCA 84 SRE creation [68] NA
PCCB 72 5¢ss creation [68] />PHEX 50 ⁄ 100 ⁄
170
5¢ss creation [128] />PKHD1 116 5¢ss creation [129] NA

PMM2 66 3¢ss creation [130] NA
PMM2 123 5¢ss creation [130,131] NA
PRPF31 175 5¢ss creation [132] NA
PTS
a
45 Branch-point
optimization
[133] NA
PTS
b
79 Py-tract
optimization
[133] NA
RB1 103 3¢ss creation [134] NA
RYR1 119 5¢ss creation [135] />SOD-1 43 5¢ss creation [136] NA
TSC2 89 5¢ss creation [137] />a
Alu-derived pseudoexons.
b
LINE-2-derived pseudoexons.
A. Dhir and E. Buratti Pseudoexons in human disease
FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS 845
actively used by evolutionary mechanisms to decrease
the probability that random activating mutations may
determine the constitutive inclusion of pseudoexon
sequences.
In this respect, one interesting molecular complex is
U1snRNP, a ribonucleoprotein complex normally
associated with 5¢ss recognition in the normal splicing
process [55]. First, U1snRNP binding to an intronic
splicing processing element has been found to inhibit

pathological pseudoexon inclusion in intron 20 of the
ATM gene (Fig. 3C). Inactivation of this element
through a four nucleotide deletion causes pseudoexon
inclusion and occurrence of ataxia telangiectasia in a
patient [56]. In a second case, binding of hnRNP E1
and U1snRNP to a weak 5¢ss efficiently silences
pseudoexon inclusion in the GHR gene [57], preventing
the development of Laron syndrome (Fig. 3D).
Finally, it should also be noted that in a variety of
pseudoexon inclusion events, the activating mutations
potentially created new splicing enhancer sequences
(Table 1). Although in very few of these cases was
trans-acting factors binding to these elements identi-
fied, in silico and experimental analyses have shown
that several of the newly created enhancer sequences
strongly correlate with potential binding to the SR
protein class of splicing regulators.
Therapeutic strategies aimed at
correcting pseudoexon inclusion in
genetic diseases
Therapeutic strategies based on antisense oligonucleo-
tide (AON) chemistry, which uses base pairing to tar-
get specific sequences in RNAs, have been extensively
employed to correct splicing disorders in human genes
[58,59]. Interestingly, apart from these therapeutic
applications, short nuclear RNAs may also play a sim-
ilar functional role to physiologically regulate exon
inclusion, such as the case of snoRNA HBII-52 in the
regulation of exon Vb inclusion in the serotonin recep-
tor 2C [60]. AONs are thought to modulate the splic-

ing pattern by steric hindrance of the recruitment of
the splicing factors to the targeted splicing competent
cis-elements, thus forcing the machinery to use the nat-
ural sites. Dominski and Kole [61] were the first to
pioneer the antisense-mediated modulation of pre-
mRNA splicing. In the earliest examples, AONs were
aimed at activated cryptic splice sites in the b-globin
and CFTR genes in order to restore normal splicing in
b-thalassaemia and cystic fibrosis patients [61,62].
Currently, however, AON strategies have been used suc-
cessfully to restore normal splicing in several disease
models.
AB
C
E
D
Fig. 2. The mutational events that determine pathological pseudoexon inclusion. The most frequent is represented by the creation
of de novo functional splice sites or branch-point elements through a single or few point mutation (A). Other mechanisms include the creation
or deletion of splicing regulatory elements (B), genomic rearrangements (C, D) and inactivation of upstream or downstream splice sites (E).
Pseudoexons in human disease A. Dhir and E. Buratti
846 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
Afibrinogenemia is caused by genetic abnormalities
within any of the three genes that encode the fibrinogen
molecule: FGA, FGB, FGG. Recently, Davis et al. [63]
showed that a homozygous c.115–600A>G point muta-
tion located deep within intron 1 of FGB causes pseudo-
exon inclusion. In this study, pseudoexon inclusion was
corrected by targeting this mutation with an antisense
phosphorodiamidate morpholino oligonucleotide.
In several forms of b-thalassaemia, two single nucle-

otide mutations (IVS2-705 and IVS2-654) in the
b-globin gene have been reported to cause pathological
pseudoexon insertion. In 1993, Dominski and Kole
[61] successfully tested 2¢-O-methylribose AONs to
restore correct splicing. Later, Sierakowska et al. [64]
also restored correct splicing and b-globin polypeptide
production using a phosphorothioate 2¢-O-methyl-
oligoribonucleotide targeted to the aberrant 3¢ss. More
recently, Gorman et al. [65,66] engineered the U7
snRNA gene to correct pre-mRNA splicing by replac-
ing the antihistone sequence with sequences targeting
b-globin aberrant splice sites (Fig. 4A).
The congenital disorders of glycosylation are caused
by defects in the PMM2 gene. Recently, Vega et al.
[130] studied a c.640–15479C>T deep intronic muta-
tion that creates a new aberrant 5¢ss in intron 7 and
caused pseudoexon activation. Antisense morpholino
oligonucleotides that targeted the aberrant 5¢ss and
3¢ss sites achieved 100% restoration of correctly
spliced mRNA.
Pseudoexon-activating mutation 3849 + 10 kb C >
T in intron 19 of the CFTR gene has been reported to
frequently cause cystic fibrosis. In their study, Fried-
man et al. [62] reported that a cocktail of 2¢-O-methyl
phosphorothioate oligoribonucleotides against different
regions of this pseudoexon abolished pseudoexon inclu-
sion and partially restored production of normal
mRNA and CFTR processed protein (Fig. 4B).
Mutations in the DMD gene are known to cause
Duchenne and Becker muscular dystrophies. Recently,

Gurvich et al. [67] demonstrated that 2¢-O-methyl
ribose phosphorothioate AONs restored normal splic-
ing in primary myoblast cultures established from two
individual patients carrying out-of-frame pseudoexon
insertion mutations (Fig. 4C).
Methylmalonic acidaemia and propionic acidaemia
are caused by different gene defects in the MUT,
A
B
C
D
Fig. 3. A schematic diagram of the tropomyosin gene with exons 2 and 3, which are mutually exclusive (exon 3 is the predominant form in
most cell types), and the nonsense exon (NE), which causes transcript degradation following its joining to exon 3 (but not exon 2). The levels
of hnRNP H ⁄ F proteins can regulate the extent of NE inclusion. (B) shows that in the NF-1 intron, 30 pseudoexon inclusion levels are regu-
lated by silencer elements in UCUU-rich motifs that bind the PTB (hnRNP I) splicing regulator. In the ATM gene, a four nucleotide deletion
(GUAA) in the intronic region between exons 20 and 21 causes the insertion of a 65 nucleotide long pseudoexon (C). Functional analysis has
demonstrated that this deletion abolished binding of an U1snRNP molecule in this position and activated a 3¢ss lying 12 nucleotides
upstream of this element. In the last case, binding of hnRNP E1 and U1snRNP to a silencer motif near a weak 5¢ss efficiently silences
pseudoexon inclusion in the GHR gene, preventing the development of Laron syndrome (D).
A. Dhir and E. Buratti Pseudoexons in human disease
FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS 847
PCCA and PCCB genes. Ugarte et al. [68] recently
reported the identification of three novel deep intronic
mutations in each of these genes that potentially lead
to pseudoexon activation through diverse mechanisms.
Antisense therapeutics using antisense morpholino
oligomers correctly restored almost complete normal
splicing that was effectively translated.
Ocular albinism type 1 involves mutations in the
OA1 gene. Vetrini et al. [69] identified a deep intronic

point mutation g.25288G>A that created a new
acceptor splice site in intron 7 of this gene and resulted
in pseudoexon inclusion. Treatment of a patient’s
melanocytes with antisense morpholino AONs comple-
mentary to the mutant sequence rescued mRNA and
protein expression levels.
Mutations in the NF-1 gene cause neurofibromatosis
type 1. Recently, Pros et al. [70] identified six neurofi-
bromatosis type 1 patients carrying three different deep
intronic mutations that create new 5¢ss leading to the
activation of the pseudoexon in the mature mRNA. In
this study, antisense morpholino oligonucleotides were
targeted against these newly created 5¢ss, effectively
restoring normal NF-1 splicing.
All of these different therapeutic strategies are sum-
marized in Table 2.
Concluding remarks
This review is part of a miniseries co-ordinated by
Diana Baralle [71] to look at emerging topics in splic-
ing research, such as the correct assessment of
sequence variants as pathogenic mutations [72]; the
development of novel splicing-based therapeutic agents
to treat HIV-1 infections [73]; and new methods in the
global analysis of alternative splicing profiles [74]. We
decided to examine the role of pseudoexons in recent
research, as no specialized reviews have appeared in
the past dealing with this particular kind of event.
From a basic science point of view, the possibility
for researchers to look at the splicing process on a
much more global scale than the single exon or the

individual gene will clarify the issues examined in this
review by helping to distinguish clearly between exons
and pseudoexons [19,75,76]. In turn, this will provide a
better appreciation regarding how the splicing process
has evolved to define ‘exons’, how it distinguishes them
from similar potentially pathological sequences (pseud-
oexons) and what is the preferential way it has chosen
to repress their recognition. In this respect, pseudoex-
on research will also provide us with an unparalleled
opportunity to understand evolutionary mechanisms
A
B
C
Fig. 4. A schematic representation of three different 5¢ss activating mutations in various disease-causing genes that activate pseudoexon
inclusion where therapeutic correction has been attempted with an antisense approach. (A) represents the IVS2-705 T>G splicing mutation
that activates a 126 nucleotide pseudoexon in intron 2 of the b-globin gene. In this case, 2¢-O-methyl ribose AONs and functionally modified
U7 snRNA were employed to block the acceptor and donor splice sites. In (B), the 3849+10kbC>T splicing mutation activates a 84 nucleo-
tide pseudoexon in intron 19 of the CFTR gene. Three 2¢-O-methyl phosphorothioate oligoribonucleotides were targeted against the splice
sites and against the pseudoexonic premature stop codon sequence to rescue normal splicing. Finally, (C) shows the c.6614+3310G>T splic-
ing mutation that activates a 137 nucleotide pseudoexon in intron 45 of the DMD gene. To restore normal splicing, 2¢-O-methyl ribose AONs
were also targeted against the donor splice site and a predicted cluster of exonic splicing enhancer sequences within the pseudoexon.
Pseudoexons in human disease A. Dhir and E. Buratti
848 FEBS Journal 277 (2010) 841–855 ª 2010 ICGEB Trieste (Italy) Journal compilation ª 2010 FEBS
that cause some of these sequences to become exons
and, of course, vice versa.
Considering that aberrant pseudoexon inclusion
events are an increasing phenomenon linked with dis-
ease, just the simple characterization of these sequences
may have some very practical consequences. The stud-
ies reported in this review clearly highlight the feasibil-

ity of using AONs to correct these types of splicing
defect (even in the absence of a complete or even par-
tial understanding of the ‘basic science’ explaining
their occurrence). From a therapeutic point of view,
the major advantage of targeting pseudoexon inclusion
events is provided by the supposition that AONs
targeted against what would normally be intronic
sequences would not be expected to remain bound to
the mature mRNA (and thus interfere with later stages
of RNA processing, such as export ⁄ translation). How-
ever, several factors will still need to be improved
before human application becomes a reality. These
start from basic studies aimed at optimizing gene ⁄ exon
specificity (that will necessarily have to be made on an
individual gene-specific basis) to the development of
appropriate carrier systems. These systems will be
absolutely necessary to achieve successful delivery, low
toxicity and avoidance of undesired immune responses.
Furthermore, even after achieving all of these aims,
there will still remain the need to optimize recurrent
administration protocols (this is an often overlooked
consideration, as none of these methods will cause a
permanent correction of mRNA splicing defects), and
determining their clearance ⁄ accumulation in human
organs ⁄ tissues. However, notwithstanding all of these
difficulties, AON technology [59,77] has already
entered the clinical trial stage for diseases such as
Duchenne muscular dystrophy (nicaltri-
als.gov) and this represents a bright hope for the not
too distant future.

Acknowledgements
This work was supported by Telethon Onlus Founda-
tion (Italy) (grant no. GGP06147) and by a European
community grant (EURASNET-LSHG-CT-2005-
518238). We thank Professor F. E. Baralle for helpful
discussion.
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Table 2. Therapeutic approaches. PMO, phosphorodiamidate morpholino oligonucleotide.
Therapeutic approaches Gene and diseases caused by pseudoexon inclusion Mode of action
Antisense PMOs FGB, afibrinogenaemia Antisense PMO specifically target the
predicted ESE motif created by the mutation
Engineered U7 snRNA HBB, b-thalassaemia Use of modified U7 snRNA to target
aberrant splice sites for long-term
restoration of correct splicing
Antisense morpholino
oligonucleotides
PMM2, congenital disorders of glycosylation;
MUT, PCCA and PCCB, methylmalonic acidaemia
and propionic acidemia; OA1, ocular albinism type 1;
NF-1, neurofibromatosis type 1
Targeting aberrant splice sites activated
due to mutations. AONs block access
of the splicing machinery to the
pseudoexonic regions in the pre-mRNAs
Antisense 2¢-O-methyl
ribose phosphorothioate
oligonucleotides;
2¢-O- methylribo-oligonucleotides
CFTR, cystic fibrosis; HBB, b-thalassaemia;
DMD, Duchenne and Becker muscular dystrophies
Targeting of antisense against aberrant
splice sites, in-frame stop codon and

predicted exonic splicing enhancers
within pseudoexons
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